This invention pertains to an improved three-dimensionally-extensive, water-swellable, matrix-like, protein-binding gel for use in solid phase immunoassay testing, and in particular, to a significant novel method of preparing such a gel.
The subject matter of the instant invention relates, more specifically, to an improvement in the method for making a gel having a material composition initially formed as described in the now co-pending, prior-filed patent application of Richard A. Harte, Anthony B. Chen and Nancy K. Kaufman, Ser. No. 483,055, filed April 7, 1983, now U.S. Pat. No. 4,540,660, patented September 10, 1985, "SUBSTRATE FOR FLUOROIMMUNOASSAY OF BIOLOGICAL FLUIDS", the entire disclosure of which is incorporated herein by reference. Also incorporated by reference in the present application, for the purpose of providing useful background information, are the disclosures of co-pending, prior-filed U.S. patent applications of Anthony B. Chen, Richard A. Harte and Nancy K. Kaufman, Ser. No. 399,855, filed July 19, 1982, now U.S. Pat. No. Des. 279,817, patented July 23, 1985, "IMMUNOASSAY TEST SLIDE", and of Richard A. Harte and John F. Hencken, Ser. No. 362,696, filed March 29, 1982, for "MULTI-LABEL IMMUNOASSAY TESTING DEVICE".
Elaborating, by way of background, the field in which the present invention has utility and importance, solid phase immunoassay techniques have been employed for about the past two decades, offering the advantage that they represent an extremely easy way for the operator to separate bound reagents (bound to a solid substrate) from reacting biochemical agents which are still in the liquid phase (Catt, K. & Treager, G. W., Science, 158, p. 1570, 1967).
While prior art solid phase techniques tend to produce sensitive, relatively simple procedures, they are prone to a serious "background noise" problem in the form of "nonspecific binding". Such binding is generally the primary cause of problems like false positive reactions, distorted quantitation values, variability, and inability to determine clearly a positive/negative cut-off value--all leading to a large "grey" zone of ambiguity which adds expense and requires frustrating retesting of samples.
In a recent review of the state of the art in solid-phase nonisotopic assays (Boraker, D. K., "SOLID PHASE MATERIALS FOR NONISOTOPIC IMMUNOASSAYS", presented at Nonisotopic Immunoassays, a one-day comprehensive conference at the New York Hilton in New York, New York, July 25, 1983), the following statement appears:
There is clearly a tendency for proteins to bind to solid phase support materials in various ways which represent non-specific binding. Such binding often occurs even with our best efforts to stop it, (Detergents, blocking proteins, etc.) to the endless frustration of those who yearn for the opportunity to push these marvelous amplification systems of enzymes and chromogenic substrates to their theoretical limits. PA1 1. Depositing on an immobilizing support structure a liquid mass of a gel-forming emulsion which, upon initial drying, is coalesceable into a thick gel-like like matrix film characterized by substantial, non-agglomerated matrix homogeneity; PA1 2. After drying of such a mass, and following coalescence of the mass into a film of the type indicated, treating the resulting film to alter matrix homogeneity by producing agglomeration in the film; and PA1 3. By such treating of the mass, establishing the object gel.
The present invention, offering a significantly improved method of producing a gel structure like that described in above-referred-to patent application Ser. No. 483,055, relates to a recently developed matrix material for use in solid-phase type immunoassays. In this material, there exists a unique micro-structure that offers a significant advance for immunoassay applications--prime among them being the almost total lack of nonspecific binding, even in the presence of undiluted body fluids.
Further explaining the background of this invention, there are, basically, three methods of attachment for binding proteins to a solid material. Over the years, an extremely large number of materials have been employed for this purpose, and reported in the literature. For example, these materials have included metal films, films of plastic such as polystyrene, polyproplyene and various acrylics, silastics and rubber-like plastics, matted fiber beds of glass wool, nylon mesh, chemically treated cellulose mats, nitrocellulose, cellulose acetates and other mixed esters of cellulose, scintered materials like clay (bentonite, kaolin, etc.), charcoal particles, treated glass surfaces, natural fibers, and many others. The three methods employed with different types of these materials are adsorption, chemical (or covalent) linkage, and gel entrapment.
Adsorption, which is the simplest method of attachment, is the most widely employed one of the three today. This kind of attachment relies on weak, electrostatic Van der Waals forces, and hydrophobic or hydrophilic bonding. Hydrogen bonding, a somewhat stronger force, may also play some part in adsorption.
Typical of the prior art adsorption procedures is the binding of specific antibody proteins to the inner walls of a plastic (polystyrene) test tube, to "fish out" specific analyte from a mix of many biochemicals in blood serum or in other body fluids. Such was first described by Catt and Treager (cited earlier herein), and became the basis of many radioimmunoassay tests developed over the following years.
A similar adsorption technique, often applied to serologies (antibody detection), has been used more recently. This technique involves allowing specific antigen solutions to stand overnight in wells of a polystyrene or polypropylene microtiter plate, permitting slow adsorption of protein to the well bottom and walls. Thereafter, the antigen solution is poured off, and the well is filled with a noninvolved protein, like bovine serum albumin, whose purpose is to cover all of the remaining binding sites on the plastic which have not electrostatically bound the antigen protein. Obviously, all of the remaining unbound sites are a major source of nonspecific binding during an assay.
A further concern of adsorption-type binding, resulting from the presence of weak electrostatic or hydrophobic forces, is the ability to break bonds during the physically strenuous activity of washing which occurs during a typical assay. Of course, washing steps after incubation of reagents is essential to remove unbound reagents. The amount of specific protein lost as a consequence of washing during an assay may vary from test well to test well, and this is a major contributing factor to poor assay precision and lack of repeatability.
Chemical or covalent bonds are much more secure, and as a consequence, the loss of reagents during testing is significantly reduced. The problems associated with covalent-type binding, however, are not trivial. For example, very few natural surfaces present exposed covalent bonding sites to protein in solution. Accordingly, it is necessary that one activate a surface, chemically or by other means (such as by plasma activation, photoactivation, etc.). In the case of chemical activation, the chemicals employed (typically periodates, carbodiimides, etc.) are generally toxic, caustic, or hazardous in other ways. Also, it is difficult to prepare a batch of material which provides consistent degrees of surface activation. Nevertheless, the advantages of covalent binding are considered to be so significant that many people take the time and trouble to perform surface treatment using these hazardous activating reagents.
The third above-mentioned method of binding, gel entrapment, typically takes place in gels with either controlled or uncontrolled pore sizes associated therewith. For example, an illustrative prior-art gel material is described in U.S. Pat. No. 3,793,445, issued to Updike and Goodfriend on February 19, 1974 for "REAGENT FOR RADIOIMMUNOASSAY", and U.S. Pat. No. 3,970,429, issued to Updike on July 20, 1976 for "METHOD FOR IMMUNOLOGIC DETERMINATION".
A general object of the present invention is to provide a method for achieving a unique gel structure which, in many ways, combines the most desirable properties of both prior art gel entrapment and covalent linkage as protein-attachment mechanisms.
More particularly, an object of the invention is to provide such a method which takes advantage of a gel structure such as that described in above-referred-to U.S. patent application Ser. No. 483,055, to enhance the same significantly with respect to its desirable binding capabilities.
As is set forth in that patent application, the gel structure therein described, without any further treatment, is known to offer a protein-binding capability which represents a significant improvement over the then-existing prior art. Experiments with such a gel, during the past few months, have shown that further treatment of the gel, in accordance with the procedure proposed by the present invention, surprisingly and dramatically improves its already excellent performance. For example, in preparing test wells containing such a gel for conducting different assays, it was noticed that, in cases where the gel was (during pre-use rehydration) exposed (immersed) for significantly longer periods of time in water, it tended to exhibit an appreciable improvement in its protein-binding ability.
Exploring further this improvement in gel characteristics, it was discovered that immersing the gel for a relatively long time period, such as up to 2-hours, produced a very noticeable enhancement of binding capabilities. It was found further that, following such a treatment, the gel retained its enhanced condition even after it was allowed to dry for use at some later time.
An alternative to prior time-extensive water soaking involved subjecting the gel to plural, relatively vigorous, but not necessarily time-extensive, water-wash and dry cycles. This kind of treatment produced a similar enhancement.
Another type of comparably useful treatment procedure, discovered later, involved the immersion of a gel of the type described in water, followed immediately by one or more freeze/thaw cycles. Here, too, similar, appreciable enhancement occurred in the gel's protein-binding ability.
Study of pre- and post-treatment gels has given a relatively clear understanding of the mechanisms of improvement that take place during each of the specific procedures which have been tried and proven. Each procedure promotes the agglomeration of particles dispersed within the gel, which particles, during preliminary gel formation, are initially distributed relatively homogeneously (with uniform, non-agglomerated dispersion) throughout the body of the gel. Such particle agglomeration selectively enlarges the porosity of gel (below its exposed surface), and this appreciably improves the free flowability of liquids in the gel during an assay, with the result that a greater population of bindable protein gets exposed to available binding sites.
A further mechanism results from the fact that the particles in the gel are interconnected by elongated polymer filaments, or strands, which are the elements that "carry" the binding sites, with the latter being distributed along the lengths of the strands. Particle agglomeration stretches many of these strands, with the consequence that adjacent binding sites distributed along a strand are moved farther away from one another. This consequence greatly minimizes steric hindrance, and makes many more binding sites functionally available for the binding of proteins.
Expressing, therefore, the method of the invention in language that embraces the several known improvement techniques and mechanisms, the same includes:
In one specific embodiment of the invention, the treating step is performed by subjecting the gel to multiple waterwash wash and dry cycles; in another specific approach, this step is performed by immersing the gel in water for a time-extended period; and in yet another procedure, the treatment step is performed by subjecting the gel to at least one freeze/thaw cycle.
The various objects and advantages attained by the invention will become more fully apparent as one reads the procedural descriptions which now follow.